Transposon being functional in mammals

ABSTRACT

Genetically modified mammalian cells comprising a Tol2 transposon transferred into a chromosome can be obtained by co-transfecting mammalian cells with a Tol2 transposase encoded by a Tol2 transposon found in medaka fish, and a Tol2 transposon lacking this transposase.

TECHNICAL FIELD

The present invention relates to transposases capable of inducingtransposition in mammals, transposons retaining these transposases,nonautonomous transposons that can be transposed in mammals, and methodsfor producing genetically modified animals using these transposases, andthe like.

BACKGROUND ART

Transposons are a powerful tool in molecular biology and geneticresearch. They are broadly used in bacteria, plants, and invertebratesfor mutagenesis, preparation of transgenic individuals, and such. On theother hand, the development of such techniques in vertebrates has beenslow. In recent years, a synthetic transposon system called “SleepingBeauty” has been constructed by connecting Tc1-like transposonfragments, which were discovered in salmonid fish (Ivics, Z., Hackett,P. B., Plasterk, R. H., & Izsvak, Z. Cell 91, 501-510 (1997)). Thetransposition of this transposon has also been confirmed in mouseembryonic stem cells and germ cell lines, etc. (Luo, G., Ivics, Z.,Izsvak, Z. & Bradley, A. Proc. Natl. Acad. Sci. USA 95, 10769-10773(1998); Yant, S. R., Meuse, L., Chiu, W., Ivics, Z., Izsvak, Z. & Kay,M. A. Nat. Genet. 25: 35-41 (2000); Fischer, S. E., Wienholds, E. &Plasterk, R. H. Proc. Natl. Acad. Sci. USA 98: 6759-6764 (2001); Horie.K., Kuroiwa, A., Ikawa, M., Okabe, M., Kondoh, G., Matsuda, Y. & Takeda,J. Proc. Natl. Acad. Sci. USA 98, 9191-9196 (2001); Dupuy, A. J. Fritz,S. & Largaespada, D. A. Genesis 30: 82-88 (2001)).

While “Sleeping Beauty” is a synthetic transposon, an active naturaltransposon has also been discovered in vertebrates. This transposon,Tol2, is the only natural transposon in vertebrates, and was cloned fromthe medaka fish (killifish) genome (Koga, A., Suzuki, M., Inagaki, H.,Bessho, Y., & Hori, H. Nature 383, 30 (1996)). Since the Tol2 sequenceis analogous to that of the Ac element in maize, it has been classifiedinto the hAT family of transposons. Furthermore, the transposition ofthis Tol2 has not only been observed in medaka fish embryos (Koga, A., &Hori, H. Genetics 156, 1243-1247 (2000)), but also in the germ line ofzebrafish (Kawakami, K., Koga, A., Hori, H., & Shima, A. Gene 225, 17-22(1998); Kawakami, K., & Shima, A. Gene 240, 239-244 (1999); Kawakami,K., Shima, A., & Kawakami, N. Proc. Natl. Acad. Sci. USA 97, 11403-11408(2000)). However, it has not been found in mammals.

Such transposons are expected to become extremely useful in both forwardgenetics, wherein genetic analysis is based on the phenotype ofmammalian cells after mutagenesis, and reverse genetics, whereinphenotype analysis is based on genes introduced during preparation oftransgenic individuals and such.

DISCLOSURE OF THE INVENTION

In order to discover functional transposons that will be powerful toolsin the gene analysis of mammals, a primary objective of the presentinvention is to analyze the activity of Tol2 in mammalian cells. Anotherobjective is to develop the likes of methods for genetic modification inmammals using this Tol2 transposon.

To achieve the above objectives, the present inventors investigatedwhether Tol2 transposon is transposable in mammals, and as a result,discovered that Tol2 transposon also functions in mammals. Inparticular, chromosomal transposition of Tol2 transposon could bedetected by incorporating a Tol2 transposase encoded by the Tol2transposon, and a Tol2 transposon lacking this transposase intodifferent vectors, and cotransfecting animals. Based on these findings,the present inventors developed the invention described below.

-   (1) A DNA encoding a transposase comprising the activity of inducing    transposition within mammalian cells, comprising the nucleotide    sequence of SEQ ID NO: 1.-   (2) A DNA encoding a transposase comprising the activity of inducing    transposition within mammalian cells, wherein said transposase DNA    is a DNA according to the following (A) or (B):    -   (A) a DNA encoding a protein comprising an amino acid sequence        in which one or more amino acids are substituted, deleted,        inserted, and/or added to the amino acid sequence of SEQ ID NO:        2, or    -   (B) a DNA hybridizing under stringent conditions with a DNA        comprising the nucleotide sequence of SEQ ID NO: 1.-   (3) A DNA complementary to a DNA comprising the nucleotide sequence    of SEQ ID NO: 1 or a complementary strand thereof, comprising a    length of at least 15 nucleotides.-   (4) A transposase encoded by the DNA of (1) or (2).-   (5) An RNA encoding the transposase of (4).-   (6) A vector comprising the DNA of (1) or (2).-   (7) A host cell comprising the DNA of (1) or (2), or the vector of    (6).-   (8) A DNA transposable in mammalian cells, wherein said DNA is a    transposon DNA according to the following (A) or (B):    -   (A) a DNA comprising the nucleotide sequence of SEQ ID NO: 3, or    -   (B) a DNA hybridizing under stringent conditions with a DNA        comprising the nucleotide sequence of SEQ ID NO: 3.-   (9) A vector comprising the DNA of (8).

(10) A host cell comprising the DNA of (8) or the vector according to(9).

-   (11) A DNA complementary to a DNA comprising the nucleotide sequence    of SEQ ID NO: 3 or a complementary strand thereof, comprising a    length of at least 15 nucleotides.-   (12) A DNA nonautonomously transposable in mammalian cells, wherein    said DNA is a nonautonomous transposon DNA comprising a deletion,    insertion, or substitution of a nucleotide sequence in a    transposase-coding region of the nucleotide sequence of SEQ ID NO:    3.-   (13) A vector comprising the nonautonomous transposon DNA of (12).-   (14) A host cell comprising the nonautonomous transposon DNA of (12)    or the vector of (13).-   (15) A kit for genetically modifying mammals, comprising the DNA    of (8) or the vector of (9).-   (16) A kit for genetically modifying mammals, comprising: the    nonautonomous transposon DNA of (12) or the vector of (13); and at    least one of the transposase DNA of (1) or (2), the vector of (6),    the transposase of (4), and the RNA of (5).-   (17) The kit for genetically modifying mammals of (16), wherein a    site to which a nucleic acid can be inserted is provided in the    nonautonomous transposon DNA of (12), or in the region of the    nonautonomous transposon DNA of (12) on the vector of (13).-   (18) A method for producing genetically modified mammalian cells,    comprising the step of introducing the DNA of (8) or the vector    of (9) into mammalian cells.-   (19) A method for producing genetically modified mammalian cells,    comprising the step of introducing mammalian cells with the    nonautonomous transposon DNA of (12) or the vector of (13); and at    least one of the transposase DNA of (1) or (2), the vector of (6),    the transposase of (4), and the RNA of (5).-   (20) The method for producing genetically modified mammalian cells    of (19), wherein a nucleic acid has been inserted in the    nonautonomous transposon DNA of (12) and the region of the    nonautonomous transposon DNA of (12) on the vector of (13).-   (21) A method for producing genetically modified mammals, comprising    the step of injecting the DNA of (8) or the vector of (9) into    nonhuman mammals.-   (22) A method for producing genetically modified mammals, comprising    the step of introducing nonhuman mammals with the nonautonomous    transposon DNA of (12) or the vector of (13); and at least one of    the transposase DNA of (1) or (2), the vector of (6), the    transposase of (4), and the RNA of (5).-   (23) A method for producing genetically modified mammals, comprising    the step generating individuals by mating a nonhuman mammal    comprising the transposase DNA of (1) or (2) with a nonhuman mammal    comprising the nonautonomous transposon DNA of (12).-   (24) A mammal for producing genetically modified nonhuman mammals,    comprising the transposase DNA of (1) or (2) or the nonautonomous    transposon DNA of (12).-   (25) The method for producing genetically modified mammals of (22),    wherein a nucleic acid has been inserted in to the nonautonomous    transposon DNA of (12), and the region of the nonautonomous    transposon DNA of (12) in the vector according to (13).

Hereinafter, the present invention will be described in detail based onembodiments.

Autonomous Transposons

The present invention relates to Tol2 transposon DNA that can betransposed in mammals. In general, the transposition of a transposonrequires an enzyme that induces transposition (a transposase) and mobileDNA sequences recognized by this transposase (transposon cis elements).The Tol2 transposons of the present invention are DNA-transposonscomprising both. These Tol2 transposons are herein referred to asautonomous Tol2 transposons because they can express transposases andtranspose themselves.

Specifically, a typical example of an autonomous Tol2 transposon DNA ofthis invention is the nucleotide sequence of SEQ ID NO: 3, however, Tol2transposon DNAs also includes DNAs that are analogous to the Tol2transposon and can be transposed in mammals in the same way. Examples ofsuch analogous transposon DNAs include DNAs that hybridize understringent conditions with DNAs comprising the nucleotide sequence of SEQID NO: 3.

Stringent hybridization conditions for isolating these DNAs that arefunctionally equivalent to the Tol2 transposon can be appropriatelyselected by those skilled in the art. In one example, afterprehybridization at 42° C. overnight in a hybridization solutioncontaining 25% formamide (50% formamide under more stringentconditions), 4×SSC, 50 mM Hepes pH 7.0, 10× Denhardt's solution, and 20ìg/ml denatured salmon sperm DNA, a labeled probe is added, andhybridization is performed by incubation at 42° C. overnight. Subsequentwashings can be performed under washing solution and temperatureconditions such as “1×SSC, 0.1% SDS, 37° C.”; more stringent conditionssuch as “0.5×SSC, 0.1% SDS, 42° C.”, and even more stringent conditionssuch as “0.2×SSC, 0.1% SDS, 65° C.”. More stringent hybridizationwashing conditions result in the isolation of DNAs that are morehomologous to the probe sequence. The aforementioned combinations of SSCand SDS concentrations and temperature conditions are mere examples, andthose skilled in the art can determine a stringency similar to thatdescribed above by appropriately combining the above-described or otherfactors that specify hybridization stringency (such as probeconcentration, probe length, and hybridization reaction time).

The Tol2 transposon discovered in medaka fish can be obtained from themedaka genomic DNA, but can be easily produced from transformants thatcomprise the Tol2 transposon, as described below. Furthermore, Tol2transposon analogues can be obtained by artificially modifying the DNAsequence of SEQ ID NO: 3, or by the above-described hybridization withDNA from species other than medaka fish.

Thus, since the Tol2 transposon and its analogues are autonomouslytransposable in mammals (mammalian cells), they are useful as tools formutagenesis, such as the random knockout of genes.

When used as a tool for mutagenesis and such, the Tol2 transposon DNAcan be carried by vectors. Vectors can be selected according to the typeof host cell, and such selection can be easily conducted by thoseskilled in the art. For example, vectors for introduction into mammaliancells may be viral vectors, nonviral vectors, or vectors that cannotreplicate in mammalian cells. Vectors comprising the Tol2 transposon donot need to autonomously replicate within mammalian cells, so long asthe Tol2 transposase encoded by Tol2 transposon can be expressed, andthe Tol2 transposon is transposable. Therefore, nonviral vectors may beused where viral vector insertion into cells is undesirable, or anarbitrary DNA sequence or chemical modification that can avoiddegradation of the Tol2 transposon DNA may be added.

In addition, the present invention includes not only the full-lengthTol2 transposon sequence, but also partial fragments thereof. Suchpartial fragments are useful as hybridization probes, PCR primers, andsuch, in analyzing the loci to which a transposon transposes aftermutagenesis in mammals. Specifically, such partial fragments arepreferably complementary to the DNA comprising the nucleotide sequenceof SEQ ID NO: 3, or to its complementary strand, and are long enough toretain specificity as probes, primers, and such, for example, 15 or morenucleotides long.

Transposases

The autonomous Tol2 transposon can itself be autonomously transposedwithin mammals. However, the transposition of Tol2 transposon can alsobe performed by supplying Tol2 transposase to nonautonomous Tol2transposons, which are transposase-deficient and unable to autonomouslytranspose.

The present invention relates to those transposases capable of inducingthe transposition of Tol2 transposon in mammals. These transposases areexemplified by, but not limited to, Tol2 transposases that comprise theamino acid sequence of SEQ ID NO: 2. For example, these transposasesalso include proteins comprising amino acid sequences analogous to theamino acid sequence of SEQ ID NO: 2, and an activity similar to that ofTol2 transposase in mammals. These analogous proteins include proteinscomprising transposase activity and an amino acid sequence in which oneor more amino acids are substituted, deleted, inserted, and/or added tothe amino acid sequence of SEQ ID NO: 2; proteins analogous to a Tol2transposase from another organism; and artificially produced Tol2transposase mutants.

The number and position of amino acid mutations in the term “amino acidsequences in which one or more amino acids are substituted, deleted,inserted, and/or added” is not limited, so long as they are within arange in which the transposition-inducing activity in Tol2 transposaseis retained. The number of amino acid mutations is typically thought tobe 10% or less of the total amino acids, preferably 5% or less, and morepreferably 1% or less.

Although Tol2 transposase can be prepared using medaka fish, it can beeasily produced using a transformant that comprises a vector thatcomprises a DNA encoding Tol2 transposase, as described below.

Furthermore, the above-described proteins, which are analogous to theTol2 transposase protein, can be prepared using hybridization techniquesknown to those skilled in the art. For example, proteins analogous tothe Tol2 transposase protein can be obtained by 1) using a DNAnucleotide sequence that encodes Tol2 transposase, e.g., the nucleotidesequence of SEQ ID NO: 1 or a part thereof, as a probe to isolate DNAshighly homologous to the Tol2 transposase cDNA from fish such as medakafish, mammals including humans, and other various species, and 2)preparing proteins from the DNAs thus isolated. Stringent hybridizationconditions for isolating such DNAs, which encode polypeptidesfunctionally equivalent to Tol2 transposase, can be appropriatelyselected by those skilled in the art. In one example, prehybridizationis carried out at 42° C. overnight, in a hybridization solutioncontaining 25% formamide (50% formamide under more stringentconditions), 4×SSC, 50 mM Hepes pH 7.0, 10× Denhardt's solution, and 20ìg/ml denatured salmon sperm DNA. A labeled probe is then added, andhybridization is carried out by warming the reaction mixture at 42° C.overnight. Subsequent washings can be performed under solution andtemperature conditions such as “1×SSC, 0.1% SDS, 37° C.”; more stringentconditions such as “0.5×SSC, 0.1% SDS, 42° C.”; or even more stringentconditions such as “0.2×SSC, 0.1% SDS, 65° C.”. The more stringent thehybridization washing conditions become, the greater the homology of theisolated DNA to the probe sequence. However, the above-describedcombinations of SSC and SDS concentrations and temperature condition aremere examples, and those skilled in the art can determine stringenciessimilar to those described above by appropriately combining theaforementioned or other factors (such as probe concentration, probelength, and hybridization reaction time), which specify hybridizationstringency.

Furthermore, it is also possible to prepare proteins analogous to Tol2transposase by isolating DNAs analogous to the Tol2 transposase DNAusing PCR (polymerase chain reaction), which is known to those skilledin the art, using the nucleotide sequence of SEQ ID NO: 1 or portionsthereof as primers, and then preparing proteins from the analogous DNAsthus isolated.

In addition, the above-described proteins analogous to Tol2 transposaseare not limited to natural proteins, and can be prepared by artificiallymodifying the Tol2 transposase protein comprising the amino acidsequence of SEQ ID NO: 2. This artificial modification can be performedby site-directed mutagenesis of a DNA encoding the Tol2 transposaseprotein, for example, the DNA of SEQ ID NO: 1, using techniques known tothose skilled in the art, such as the deletion-mutant preparationmethod, PCR method, and cassette mutation method.

Whether the obtained proteins, which are analogous to the Tol2transposase, comprise a transposition-inducing activity similar to thatof Tol2 transposase, can be assessed by analysis. This analysis can beperformed according to the Example described below.

Transposase DNAs

DNAs encoding transposases of the present invention (hereinafterreferred to as “transposase DNAs”) include cDNAs, genomic DNAs, andsynthetic DNAs, so long as they are DNAs that encode the activity ofinducing transposition.

A preferred example of a cDNA that encodes a transposase of thisinvention is a cDNA that encodes the above-described Tol2 transposase,specifically, a DNA of SEQ ID NO: 1. In addition to this cDNA, cDNAscomprising an activity similar to that of Tol2 transposase are alsoincluded. Such cDNAs can be screened from cDNA libraries derived frombiological tissues in which proteins comprising the aforementionedactivity are expressed. Screening can be carried out by labeling DNAs ofSEQ ID NO: 1, their fragments, or the like, and using these as probesfor hybridization. Alternatively, the above-described cDNAs may beprepared by RT-PCR by using synthetic oligonucleotides that compriseportions of a DNA of SEQ ID NO: 1 as primers, and with an RNA templatederived from tissues expressing proteins that comprise an aforementionedactivity.

A preferred example of a genomic DNA that encodes a transposase of thepresent invention is a DNA of SEQ ID NO: 3. Since this genomic DNA hasbeen identified in medaka fish, it can be obtained from the total DNA ofmedaka fish, its genomic DNA library, animal genomes such as that ofzebrafish into which the transposon has been already introduced, or thelike. Furthermore, in addition to the genomic DNAs of SEQ ID NO: 3,genomic DNAs comprising an activity similar to that of Tol2 transposaseare also included. Such genomic DNAs may be obtained in the same way asin the above-described preparation of cDNAs, using labeled DNAs of SEQID NO: 1 or 3, their fragments, or the like, as probes, and carrying outhybridization from genomic DNA libraries derived from biologicaltissues. Alternatively, they may be prepared by RT-PCR using syntheticoligonucleotides comprising a portion of a DNA of SEQ ID NO: 1 or 3 asprimers, and with a template of RNA derived from biological tissue.

Furthermore, the aforementioned cDNAs and genomic DNAs can also besynthesized using a commercially available DNA synthesizer. For example,they can be prepared by synthesizing a DNA of SEQ ID NO: 1 or 3 and itscomplementary strand, and then annealing these to form a double strand.

Since the above-described transposase DNAs encode transposases thatcomprise the activity of inducing transposition within mammalian cells,these DNAs are not only useful as materials for producing thesetransposases, but can also be used to introduce transposase DNAsdirectly into mammals, to express the transposases within cells.

The present invention relates to vectors comprising the transposaseDNAs. In order to produce the transposases and express them withinmammals using the transposase DNAs, as described above, theaforementioned transposase DNAs are preferably incorporated into desiredexpression vectors. Vectors that can be used herein are not particularlylimited, and can be appropriately selected from expression vectors knownto those skilled in the art, according to the host in to which a vectorcomprising a transposase DNA is to be introduced, its usage, and thelike. Examples of vectors for expressing transposases in mammals(mammalian cells) include viral vectors such as retroviral vectors,adenoviral vectors, adeno-associated viral vectors, vaccinia viralvectors, lentiviral vectors, herpes viral vectors, alphaviral vectors,EB viral vectors, papilloma viral vectors, and foamy viral vectors; andnonviral vectors such as cationic liposomes, ligand DNA complexes, andgene guns (Y. Niitsu et al. Molecular Medicine 35: 1385-1395 (1998)).

The present invention also includes partial fragments of the transposaseDNAs. The above transposase DNAs can be used as total sequences toexpress the transposases and such, and their partial fragments are alsouseful as hybridization probes, PCR primers, or ribozyme derivatives.Therefore, for these purposes, these partial fragments are preferablylong enough to maintain specificity as a probe, and such. For example,these fragments can be 15 nucleotides or longer. Examples of suchpolynucleotides are those that specifically hybridize with DNAscomprising the nucleotide sequence of SEQ ID NO: 1 or 3, orcomplementary strands thereof. Herein, “specifically hybridizing” meansthat no significant cross hybridization occurs with DNAs that encodeother proteins. The above-described probes and primers can be used incloning DNAs encoding the present protein, and in detecting the presentDNAs.

Nucleic acids capable of expressing the transposases of the presentinvention include not only the above-described DNAs, but also RNAs. RNAsmediate transposase expression from the transposase DNAs. Therefore,transposases can also be expressed within mammalian cells by introducingmammalian cells with transposase RNAs instead of DNAs. Such RNAs can beprepared by transcribing the above-described transposase cDNAs orgenomic DNAs, or by using an RNA synthesizer to synthesize RNAs thatcorrespond to the above-described transposase cDNAs.

Nonautonomous Tol2 Transposons

Another embodiment of the present invention relates to nonautonomousTol2 transposons, which are not autonomously transposable in mammalssince they lack transposase, and which are transposable on supply ofTol2 transposase.

Tol2 transposons are autonomously transposable. Therefore, after a Tol2transposon transfers into a mammalian chromosome, it may be excised andtransfer to another region. Although such autonomous transposons can actalone to introduce mutations into mammalian chromosomes, they areunstable in terms of the possibility of transposition from a transferredposition to another position. Therefore, by deleting the transposasefrom the Tol2 transposon, the ability to autonomously transfer isdeleted, and re-transfer after transfer to a chromosome can besuppressed. Thus, mutations can be stably introduced into chromosomes,and genes can be modified. Such nonautonomous Tol2 transposons includeDNAs which have lost their transposase activity due to deletion,insertion, or substitution of a nucleotide sequence in a region encodingthe transposase of the above-described transposon DNAs, which comprisethe nucleotide sequence of SEQ ID NO: 3, or analogous DNAs comprisingequivalent functions.

This deletion, insertion, or substitution of a nucleotide sequence in toa region encoding a transposase is not limited as to the number or typeof nucleotides and such in the varied region, so long as it is amutation that enables loss of transposase activity. It is not limited tomutations in regions encoding a transposase, and also includes mutationsthat can inhibit transposase expression by a mutation such as deletion,insertion, and substitution in a non-coding region. However, thesemutations are limited to those whereby cis elements for transpositionare not deficient, to ensure the ability to transfer as a transposon.Such cis elements for transposition can be identified by preparing aseries in which the nucleotide sequence of SEQ ID NO: 3 issystematically deleted, using methods known to those skilled in the art,commercially available deletion kits, or the like, and then analyzingregions necessary for transfer.

One example of a deletion mutation in a transposase coding region isthat comprising a deletion from 5′ side of exon 2 to around the centerof exon 4 in Tol2 transposon DNA, as shown in FIG. 1 (specifically,nucleotide 2230 to nucleotide 4146 in SEQ ID NO: 3), but a deletionlonger or shorter than this may be used, and the deleted region may bedivided into a number of sections. One example of an insertion mutationis a mutation that deletes transposase activity by inserting a linkersequence, restriction enzyme recognition sequence, multicloning site, orarbitrary gene into a transposase-encoding region of the Tol2transposon, or the like. One example of a substitution mutation is thatin which a nucleotide sequence in a transposase-encoding region or thelike is substituted with another nucleotide sequence, deleting thetransposase's transposition-inducing activity. These deletion,insertion, and substitution mutations may be used singly or incombination. One example of such a combination of mutations isnonautonomous transposon DNA comprising the nucleotide sequence of SEQID NO: 4. This DNA comprises a deletion of nucleotides 2230 to 4146 inthe Tol2 transposon DNA of SEQ ID NO: 3, and the insertion into thisdeleted region of a linker sequence comprising a XhoI recognitionsequence comprising “AGATCTCATATGCTCGAGGGCCC”.

Such nonautonomous Tol2 transposon DNAs do not themselves comprisetransposase activity. Therefore, when introducing a mutation into achromosome using such DNAs, the DNAs can be stably maintained on thechromosome unless a transposase is supplied from outside. Such mutationintroduction is therefore advantageous in identifying functional genesand producing stable transgenic individuals.

Further, the present invention relates to vectors comprising theabove-mentioned nonautonomous Tol2 transposon DNAs. While nonautonomousTol2 transposon DNAs can be used as they are, they can also be connectedto vectors, for example, to prevent end portion degradation whenintroduced into a mammal or mammalian cell. These vectors can beselected depending on the host to be used or their purpose. For example,vectors for introducing these DNAs into mammals (or their cells) can beany of the virus-based vectors (e.g., adenovirus vectors,adeno-associated virus vectors, vaccinia virus vectors, lentivirusvectors, herpes virus vectors, alpha virus vectors, EB virus vectors,papilloma virus vectors, foamy virus vectors, and retrovirus vectors)and nonviral vectors (e.g., cationic liposomes, ligand DNA complexes,and gene guns) (Y. Niitsu et al., Molecular Medicine 35: 1385-1395(1998)).

Further, the present invention relates to mammalian cells comprising theabove-mentioned nonautonomous Tol2 transposon DNAs or vectors comprisingthe same. Since the nonautonomous Tol2 transposon DNAs in thesemammalian cells can transfer and introduce mutations on supply oftransposase, such cells comprising the DNAs or vectors can be used toanalyze functional genes, manufacture knockout animals, and the like.The host mammalian cells for analysis of functional genes are notparticularly restricted, and may be cells derived from primates such ashumans, or from rodents such as mice. These mammalian cells may besomatic cells or reproductive cells, and the somatic cells include cellsderived from various organs. In the production of knockout animals,cells comprising a reproductive ability, such as germ cells, arepreferable for use as the host mammalian cells. In such cells, theabove-mentioned DNA or vector may be maintained outside of a chromosomeor inside of a chromosome.

Systems for Producing Mutant Mammalian Cells

Another embodiment of the present invention relates to systems formodifying mammalian genes. These systems for modifying mammalian genesare systems of modifying mammalian chromosomes using the above-mentionedautonomous or nonautonomous Tol2 transposons. Such “modifications”include knockdown mutations that break existing genes on chromosomes,transgenic mutations that insert novel genes, and modificationsincluding combinations of both.

The first example of a knockdown mutation is a system comprising theabove-mentioned autonomous Tol2 transposon DNA, as typified by thenucleotide sequence of SEQ ID NO: 3, or a vector comprising this DNA. Asdescribed above, autonomous Tol2 transposon DNAs maintain a transposaseand can transfer autonomously in mammalian cells. Therefore, theabove-mentioned DNAs or vectors can be introduced into mammalian cellsfor random gene knockdowns and the like.

A second knockdown mutation system is a system for modifying a mammaliangene that comprises a nonautonomous Tol2 transposon DNA, such as thenucleotide sequence of SEQ ID NO: 4 or a vector comprising this, and atleast one of the Tol2 transposase DNAs typified by the nucleotidesequence of SEQ ID NO: 1, a vector comprising this DNA, a transposase astypified by the amino acid sequence of SEQ ID NO: 2, and a RNA encodingthis transposase. That is, nonautonomous Tol2 transposon DNAs themselvesare not transposable since they lack transposase. However, by supplyingmammals with transposase DNAs or RNAs, or directly supplying transposaseproteins together with these nonautonomous Tol2 transposon DNAs, theTol2 transposase DNAs can transfer to chromosomes in the mammals tointroduce mutations into random genes on the chromosomes. When a DNA isused as a source for supplying Tol2 transposase, this transposase DNAmay be carried by entities other than those carrying the nonautonomousTol2 transposon DNAs, i.e., separate fragments or separate vectors, orboth DNAs may be on the same vector.

A system for transgenic mutation can be the second knockdown mutationsystem, mentioned above. Namely, a system comprising nonautonomous Tol2transposon DNAs or vectors comprising such DNAs, and a Tol2 transposasesupply source (e.g., DNAs, RNAs, proteins, etc.). For easy use of suchsystems as systems for transgenic mutation, the above-mentionednonautonomous transposon DNAs or the nonautonomous transposon DNAregions on the vectors comprising these DNAs preferably comprise a siteinto which an arbitrary nucleic acid can be inserted. Herein, “anarbitrary nucleic acid” is a nucleic acid whose insertion into amammalian chromosome is desired, and, for example, genes whose functionis to be analyzed can be arbitrarily selected. To facilitate thisincorporation of arbitrary nucleic acids into nonautonomous transposonDNAs, a restriction enzyme recognition sequence can be provided as “asite into which an arbitrary nucleic acid can be inserted”. Suchrestriction enzyme recognition sequences may be recognition sites forone restriction enzyme, and may preferably be a multicloning site thatcomprises recognition sites for a plurality of restriction enzymes. Byproviding such cloning sites as described above, genes for analysis canbe easily inserted into nonautonomous transposon DNAs. By introducing amammalian cell with a nonautonomous transposon DNA that comprises anarbitrary nucleic acid, along with a Tol2 transposase supplying source(a Tol2 transposase DNA, a vector comprising this, a Tol2 transposaseRNA, or a Tol2 transposase protein, transfer of the nonautonomous Tol2transposon DNA to a chromosome, and efficient incorporation of thedesired nucleic acid into the chromosome can be achieved. In suchtransfers of Tol2 transposons into chromosomes, DNAs other than thetransposon DNA, such as unnecessary DNAs like vector sequences, are nottransferred, even if the Tol2 transposon DNA has been incorporated intoa vector. Therefore, these transgenic mutation systems are expected tobe useful not only for production of transgenic animals and the like,but also as safe systems for gene therapy. That is, they may also beutilized medically as systems for supplementing disease-causing genesand the like.

Methods for Producing Mutant Mammalian Cells

Further, the present invention relates to methods for producing mutantmammalian cells using Tol2 transposon DNAs. A first method for producingmutant mammalian cells using Tol2 transposon DNA is a method comprisinga process of introducing mammalian cells with an autonomous Tol2transposon DNA, as typified by SEQ ID NO: 3, or a vector comprisingsuch.

The target cells of such methods are not particularly limited s to theirspecies and the like, so long as they are mammalian cells. The targetcells thus include cells derived from primates such as humans, and cellsderived from rodents such as mice. Methods for introducing theabove-mentioned transposon DNAs into such cells are not particularlyrestricted, and include liposome methods, electroporation methods,calcium phosphate methods, and gene gun methods. For germ cells and thelike, microinjection methods and such can be used.

Introducing autonomous Tol2 transposon DNAs into target mammalian cellsin this way enables transposition of Tol2 transposon DNAs intochromosomes by the action of transposases encoded by the autonomous Tol2transposon DNAs in the cells, thus producing mutant mammalian cells.

A second method for producing mutant mammalian cells is a methodcomprising a step of introducing the cells of a non-human mammal with annonautonomous Tol2 transposon DNA, such as the nucleotide sequence ofSEQ ID NO: 4, or a vector comprising such, and at least one of a Tol2transposase DNA as typified by the nucleotide sequence of SEQ ID NO: 1,a vector comprising this DNA, a transposase typified by the amino acidsequence of SEQ ID NO: 2, and a RNA encoding this transposase.

Since the above-mentioned first method may cause re-transfer of Tol2transposases in the mutant mammalian cells produced, the second methodusing a nonautonomous Tol2 transposon is preferred for reducing thepossibility of re-transfer. Also in the second production method, thetarget mammalian cells are not as restricted as in the above-mentionedfirst production method.

In the methods for introducing a Tol2 transposon DNA along with a sourcefor supplying a transposase to target cells, such as a Tol2 transposaseDNA, a vector comprising this DNA or a Tol2 transposase RNA,co-transfection may be performed using the various methods described forthe first production method (e.g., liposome methods, electroporationmethods, calcium phosphate methods, gene gun methods, and microinjectionmethods). When a Tol2 transposase DNA is used as a transposase supplysource, it can be subjected to the above-mentioned co-transfection in afragment or vector separate from that of the nonautonomous Tol2transposon DNA. Alternatively, both DNAs can be incorporated into thesame vector for introduction to mammalian cells by any of theabove-mentioned methods. When a Tol2 transposase protein is directlyused as a Tol2 transposase supply source, the protein may be suppliedsimultaneously in transfecting the above-mentioned nonautonomous Tol2transposon DNA into target cells by microinjection, or it may besupplied by endocytosis.

The above-mentioned methods for producing mutant mammalian cells canmainly be utilized as methods capable of transferring Tol2 transposonsinto chromosomes to produce cells in which a gene or the like israndomly knocked out. The cells thus produced can be used as tools forforward genetics. That is, the phenotype of the cells may change due tothe destruction of some gene. Therefore, by analyzing the phenotypes ofcells produced by such methods, and identifying gene loci into whichtransposon DNAs have been inserted in cells that comprise alteredphenotypes, it is possible to screen for functional genes. The analysisof phenotypes includes not only analysis of expression on the surface ofcells but also analysis of wide kinetic change in cells. Identificationof a locus into which a transposon DNA has been inserted can beperformed by, for example, hybridization using the transposon DNA usedfor introducing the mutation, or its portion, as a probe.

The methods for producing mutant mammalian cells of the presentinvention can be used not only as methods for producing random knockoutmammalian cells as described above, but also for producing transgeniccells into which a nucleic acid such as a desired gene has beenintroduced. When producing such cells into which a desired exogenousgene or the like has been introduced, a desired nucleic acid such as agene whose function is to be analyzed is pre-inserted into anabove-mentioned nonautonomous Tol2 transposon DNA, or a nonautonomoustransposon DNA region on a vector comprising the same. Suchnonautonomous Tol2 transposon DNA or vector comprising the same, intowhich a desired nucleic acid has been inserted, is then introduced intotarget mammalian cells. An above-mentioned Tol2 transposase supplysource (Tol2 transposase DNAs, vectors comprising these DNAs, Tol2transposase RNAs, or Tol2 transposase proteins) is simultaneouslyprovided to the cells along with the introduction of a nonautonomousTol2 transposon DNA, or are provided at different times. By the actionof this Tol2 transposase, the Tol2 transposon DNA comprising the desirednucleic acid transfers to a chromosome in the cells, and the desirednucleic acid is efficiently inserted into the chromosome. Thus, by usinga transposon of the present invention as a carrier for transporting adesired gene to a chromosome, desired nucleic acids, such as genes andDNAs whose functions are to be investigated, can be efficiently insertedinto chromosomes. Cells produced here can be used as material forreverse genetics. That is, if introducing a cell chromosome with atransposon DNA and a desired nucleic acid causes a change in cellphenotype, the function of this desired nucleic acid can be analyzed byanalyzing this phenotype.

Methods for Producing Genetically Modified Animals

Another embodiment of the present invention relates to methods forproducing genetically modified animals using Tol2 transposons.

A first method for producing genetically modified animals is a methodcomprising a step of injecting a non-human mammal with an autonomousTol2 transposon DNA, typified by the nucleotide sequence of SEQ ID NO:3, or a vector comprising the same.

The target mammals of these methods include primates such as monkeys,and rodents such as mice, so long as they are non-human mammals. Asmethods for introducing these mammals with the above-mentionedtransposon DNAs or vectors, transport into an animal body can be byinjection that is intravascular, intramuscular, subcutaneous, or thelike. For example, in the case of a mouse, its tail is cut and theabove-mentioned DNA or the like can be injected through a tail vein.Thus, DNAs injected in this way are distributed into amammal's tissuesand the like, and reach the somatic cells, reproductive cells, and soon. The Tol2 transposon DNA transfers to a chromosome in any of thesecells, and a mammal comprising a chromosomal mutation can thus beproduced. When the above-mentioned transposon DNA transfers to achromosome in a reproductive cell, this transposon DNA can betransferred from the generated mammal to its descendents, producing manymutant mammals.

A second method is a method comprising a step of introducing a non-humanmammal with a nonautonomous Tol2 transposon DNA, such as a nucleotidesequence of SEQ ID NO: 3 or a vector comprising the same, and at leastone of a Tol2 transposase DNA typified by the nucleotide sequence of SEQID NO: 1, a vector comprising this DNA, a Tol2 transposase typified bythe amino acid sequence of SEQ ID NO: 2, and a Tol2 transposase RNAencoding the same.

The target animals for this second method are also non-human mammals.Injection of mammals with these nonautonomous Tol2 transposon DNAs orthe like, and a transposase supply source (Tol2 transposase DNAs,vectors comprising such DNAs, Tol2 transposases, or Tol2 transposaseRNAs) can be performed by intravascular injection or the like, as in thefirst method above. Nonautonomous Tol2 transposon DNAs or vectors may beinjected simultaneously with the above-mentioned transposase supplysources, or one may be injected prior to the other.

Thus, by injecting mammalian cells with transposase supply sources andnonautonomous Tol2 transposon DNAs or the like, these injected materialsare distributed to the mammal's tissues and the like, and reach somaticcells, reproductive cells, and so on. The transposases act on thenonautonomous Tol2 transposon DNAs in such cells, transferring thenonautonomous Tol2 transposon DNA to a chromosome and producing mammalsthat comprise chromosomal mutations. Note that in such cases, when theabove-mentioned transposon DNAs transfer to chromosomes in reproductivecells, the transposon DNAs can be transferred from the generated mammalto its descendents, and many mutant mammals can be produced.

A third method is a variation of the second method, comprising a step ofgenerating individuals by mating a nonhuman mammal comprising a Tol2transposase DNA as typified by the nucleotide sequence of SEQ ID NO: 1,with a nonhuman mammal comprising a nonautonomous Tol2 transposon DNA,such as the nucleotide sequence of SEQ ID NO: 4. Thus, this method isdifferent from the second method of injecting a Tol2 transposase supplysource and a nonautonomous Tol2 transposon DNA into one mammal, in thata mammal previously comprising a nonautonomous Tol2 transposon DNA and amammal comprising a transposase DNA are mated to transfer both DNAs intooffspring. Thus, to transfer these DNAs to offspring, the nonautonomousTol2 transposon DNAs and Tol2 transposase DNAs must be maintained in thereproductive cells of the respective mammals. In addition, since matingis prerequisite to this method, both mammals must be of different sex.

Parent mammals comprising an autonomous Tol2 transposon DNA may beproduced by the above-mentioned second method; or generated from germcells or the like comprising autonomous Tol2 transposon DNAs; orproduced without the action of a Tol2 transposase by usual methods forproducing transgenic individuals, that integrate DNAs into chromosomes.The other parent mammals, comprising Tol2 transposase DNAs, can beproduced by usual methods for producing transgenic individuals(“Manipulating the mouse embryo, a laboratory manual”, second edition,Kindai Syuppan, Brigid Hogan et al., translated by Kazuya Yamauchi etal.).

The above three methods for producing genetically modified mammals canbe used mainly for the purpose of producing knockout individuals inwhich a mutation has been introduced into a chromosome. Accordingly,mammalian cells produced by these methods can be used to screen novelfunctional genes. That is, the phenotypes of mutant mammals producedherein are analyzed, and animals comprising mutant phenotypes areselected. These phenotypes may be systemic, or specific to certainorgans or tissues. Functional genes may also be screened by identifyingthe tissue, chromosomal position, and such, in which transposon DNA ispresent in animals thus selected.

The methods for producing genetically modified animals of the presentinvention can be utilized not only as methods for producing random geneknockout animals, as described above, but also as methods for producingtransgenic animals into which desired nucleic acids have beenintroduced. When carrying out the methods of the present invention forsuch purposes, a desired nucleic acid is pre-inserted into anabove-described nonautonomous Tol2 transposon DNA, or a nonautonomoustransposon DNA region on a vector. The above-mentioned second productionmethod of the present invention is then performed using a nonautonomoustransposon DNA into which the desired nucleic acid has been inserted, ora vector comprising the same. That is, a nonautonomous Tol2 transposonDNA into which a desired nucleic acid has been inserted, or the vectorcomprising this, is injected into a mammal. In this injection, totransfer a nonautonomous Tol2 transposon, a source of a Tol2 transposase(Tol2 transposase DNAs, vectors comprising these, Tol2 transposase RNAs,or Tol2 transposase proteins) is injected simultaneously or separatelyinto the mammal. In this mammal, the action of the transposase transfersthe nonautonomous transposon DNA into a chromosome, and this transfer ofnonautonomous transposon DNA produces a mutant mammal that comprises anarbitrary nucleic acid introduced into a chromosome. According to thepresent methods, a desired nucleic acid can be efficiently introducedinto a mammal using the function of a transposon. In addition, by usingthese methods, a desired nucleic acid can be efficiently introduced intoa mammal to impart a desired function. Furthermore, these methods canalso be used as methods for analyzing the function of a gene, by placinga desired nucleic acid, such as a test nucleic acid whose function is tobe analyzed, on to a transposon and producing mutant mammals by apresent method, and analyzing the phenotype of the produced animals.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the structure of Tol2, pT2 KPKneo,and a transposase expression vector. A Tol2 transposase mRNA istranscribed from a transposase DNA comprising three of the introns onthe Tol2 transposon. pT2 KPKneo has a structure whereby a part of Tol2is substituted with a PGK-neo cassette (the dotted lines), and has threeBglII recognition sites. pCAGGS-T2 TP is constructed by cloning Tol2cDNA into pCAGGS (Niwa, H., Yamamura, K. & Miyazaki, J. Gene 108,193-200 (1991)). The black line and black arrowheads above pT2 KPKneorespectively show the probe and primers used in this analysis.

FIG. 2 shows the results of analyzing genomic DNA extracted from G418resistant ES cell clones transformed using pT2 KPKneo and pCAGGS-T2 TP(clones #1 to #10), and from G418 resistant ES cell clones transformedusing pT2 KPKneo and PCAGGS (clones #11 to #20). (a) photographs showingthe results of Southern blot analysis of genomic DNA after digestionwith BglII using the probe shown in FIG. 1. (b) photographs showing theresults of PCR analysis using the f20 and ex4f primers (upper panel),and the PGKr1 and r7 primers (lower panel). PCR using the PGKr1 and r7primers was performed to confirm the presence of Tol2 DNA in the DNAsamples. In the drawing, M represents a marker, E representsnon-transformed ES cell DNA, P represents pT2 KPKneo plasmid DNA, and“-” represents a blank (no DNA). (c) a diagram showing the DNA sequencesat integration sites of clones 1, 9, and 10. In these sequences, theTol2 sequences are italicized, and the eight base forward repeatingsequences are underlined.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in more detail with reference toan Example, but is not to be construed as being limited thereto.

EXAMPLE 1 Analysis of Transposition of Tol2 in Mammal

To develop novel transposons that can be used in mammals, Tol2's abilityto transpose in mammalian cells was analyzed using the following2-component analysis system:

Two plasmids were used in the 2-component analysis system: pT2 KPKneoprovided with a cis element of Tol2, and pCAGGS-T2TP provided with atrans element of Tol2 (FIG. 1). First, plasmid pT2 KPKneo wasconstructed as follows: pTol2-tyr (Kawakami, K., Shima, A., &Kawakami,N. Proc. Natl. Acad. Sci. USA 97, 11403-11408. (2000)) was modified toconstruct a nonautonomous transposon DNA (SEQ ID NO: 4) by connectingthe region from 1 to 2229 and the region from 4147 to 4682 of thefull-length sequence of Tol2 (SEQ ID NO: 3), through a linker sequencecomprising a XhoI recognition sequence. This DNA was incorporated intothe pCAGGS plasmid (Niwa, H., Yamamura, K. & Miyazaki, J. Gene 108,193-200 (1991)) to construct a nonautonomous Tol2 transposon vector. Tointroduce a drug-resistant marker into the above vector, a PGK-neocassette (a cassette comprising a neomycin-resistant gene connecteddownstream of a PGK promoter, and PGK polyadenylation signal sequencefurther downstream of this) was inserted into XhoI in theabove-mentioned nonautonomous transposon DNA. pT2 KPKneo was thusconstructed. Another plasmid pCAGGS-T2TP was constructed byincorporating Tol2 cDNA (SEQ ID NO: 1) into pCAGGS.

The above-mentioned pT2 KPKneo (50 μg) was introduced into mouse EScells by electroporation, together with pCAGGS-T2TP (300 μg) or pCAGGSvector carrying no transposase (300 μg). After electroporation, cellswere plated on several dishes, and cultured using a standard ES cellculturing method in the presence of G418 (175 μg/ml). In theco-transfection of pT2 KPKneo and pCAGGS-T2TP, a total number of about1.1×10⁴ G418-resistant (G418^(R)) colonies were obtained. On the otherhand, in the co-transfection of pT2 KPKneo and pCAGGS vector, aboutfifty G418^(R) colonies were obtained. That is, the transformationefficiency when co-transfecting with pCAGGS-T2TP was about 200 timesthat when using pCAGGS vector. This result showed that the transposaseexpressed from pCAGGS-T2TP exerts a positive effect on transformationefficiency.

To analyze whether or not this high efficiency transformation occurredbecause of chromosomal integration by the transposition of the transfercassette, ten G418-resistant colonies obtained in each of the abovetransfection experiments were isolated and cultured, genomic DNA wasextracted from each colony, and various analyses were performed.Southern blot analysis of the genomic DNA showed that theabove-mentioned transfer cassette was inserted into a chromosome inthese G418-resistant ES cell colonies (FIG. 2 a). The chromosomallyinserted sequence was then analyzed by PCR using primers f20(5′-TTTACTCAAGTAAGATTCTAG-3′ (SEQ ID NO: 0.5)) and ex4f(5′-GCTACTACATGGTGCCATTCCT-3′ (SEQ ID NO: 6)), as shown in FIG. 1. FromES colonies obtained by co-transfection using pCAGGS-T2TP (FIG. 2 b,upper panel, lanes 1 to 10), an amplified DNA band was not detected. Onthe other hand, from ES colonies obtained by co-transfection usingpCAGGS (FIG. 2 b, upper panel, lanes 11 to 20), about 200 bp DNA bandwas amplified. The results of this PCR showed that, in the ES clonesco-transfected with pCAGGS-T2TP, only the transfer cassette was insertedinto the ES clone, and an adjacent vector sequence was not inserted.

Finally, to analyze DNA fragments comprising connecting portions of theinserted Tol2 sequence and adjacent mouse genomic sequences, inverse PCRwas conducted on three ES clones co-transfected with pCAGGS-T2TP. Theamplified fragments were cloned and sequenced. The three ES clones usedhere are clones in which insertion of a single Tol2 sequence wasdetected using Southern blotting (FIG. 2 a, lanes 1, 9, and 10). In eachof these three clones, the Tol2 insert was surrounded by mousechromosomal DNA and an 8 bp forward repeat sequence. This sequence wasalways discovered and formed in Tol2 target sites (FIG. 2 c). Wild typeDNA from the above region prior to transfer of Tol2 was cloned fromuntransfected ES cells. Each region comprised one copy of the 8 bpsequence. These results showed that the transposase produced bypCAGGS-T2TP functioned in mouse ES cells, and by the action of thistransposase, a nonautonomous Tol2 element was transferred and insertedinto a chromosome. In clone #10, a partial genomic sequence comprisingthe Tol2 insert region coincided completely with a mouse EST sequence(BB629503) (118 bp/118 bp), and the end of this homology terminated atGT on the chromosome sequence. This suggests the transfer and insertionof Tol2 into an exon in this clone.

Tol2 was shown to be able to transfer not only in fish but also in mice,which are mammals. Although host factors correlated with the Tol2transposition reaction are not known, such factors may be commonlypresent in these hosts.

Tol2 belongs to the transposon hAT family, which is different from theTcl/mariner family to which Sleeping Beauty belongs. This means thatboth transposon systems may have different properties such as transferefficiency, preferred transfer sequences, and the like. In fact,although Sleeping Beauty usually transfers to TA sequences (Ivics, Z.,Hackett, P. B., Plasterk, R. H., & Izsvak, Z. Cell 91,501-510 (1997)),such specificity was not observed in Tol2 (FIG. 2C, Koga, A., Suzuki,M., Inagaki, H., Bessho, Y., & Hori, H. Nature 383, 30 (1996), Kawakami,K., Shima, A., & Kawakami, N. Proc. Natl. Acad. Sci. USA 97, 11403-11408(2000), Koga, A., & Hori, H. Genetics 156, 1243-1247 (2000)). Therefore,Tol2 can be used as a novel tool for methods of producing transgenicmammals and methods of introducing mutations.

Industrial Applicability

The present invention provides novel transposons that function inmammals. Such transposons can be utilized as autonomous transposonscomprising a transposase, or nonautonomous transposons with a separatetransposase. Such transposons can be powerful tools in producingknockout animals and transgenic animals. The present invention alsoprovides methods for modifying mammals using these transposons and thelike. These methods are expected to be significantly useful in analyzingthe function of various mammalian genes, and in discovering functionalgenes.

1. A DNA encoding a transposase comprising the activity of inducingtransposition within mammalian cells, comprising the nucleotide sequenceof SEQ ID NO:
 1. 2. A DNA encoding a transposase comprising the activityof inducing transposition within mammalian cells, wherein saidtransposase DNA is a DNA according to the following (A) or (B): (A) aDNA encoding a protein comprising an amino acid sequence in which one ormore amino acids are substituted, deleted, inserted, and/or added to theamino acid sequence of SEQ ID NO: 2, or (B) a DNA hybridizing understringent conditions with a DNA comprising the nucleotide sequence ofSEQ ID NO:
 1. 3. A DNA complementary to a DNA comprising the nucleotidesequence of SEQ ID NO: 1 or a complementary strand thereof, comprising alength of at least 15 nucleotides.
 4. A transposase encoded by the DNAof claim 1 or
 2. 5. An RNA encoding the transposase of claim
 4. 6. Avector comprising the DNA of claim 1 or
 2. 7. A host cell comprising theDNA of claim 1 or 2, or the vector of claim
 6. 8. A DNA transposable inmammalian cells, wherein said DNA is a transposon DNA according to thefollowing (A) or (B): (A) a DNA comprising the nucleotide sequence ofSEQ ID NO: 3, or (B) a DNA hybridizing under stringent conditions with aDNA comprising the nucleotide sequence of SEQ ID NO:
 3. 9. A vectorcomprising the DNA of claim
 8. 10. A host cell comprising the DNA ofclaim 8 or the vector according to claim
 9. 11. A DNA complementary to aDNA comprising the nucleotide sequence of SEQ ID NO: 3 or acomplementary strand thereof, comprising a length of at least 15nucleotides.
 12. A DNA nonautonomously transposable in mammalian cells,wherein said DNA is a nonautonomous transposon DNA comprising adeletion, insertion, or substitution of a nucleotide sequence in atransposase-coding region of the nucleotide sequence of SEQ ID NO: 3.13. A vector comprising the nonautonomous transposon DNA of claim 12.14. A host cell comprising the nonautonomous transposon DNA of claim 12or the vector of claim
 13. 15. A kit for genetically modifying mammals,comprising the DNA of claim 8 or the vector of claim
 9. 16. A kit forgenetically modifying mammals, comprising: the nonautonomous transposonDNA of claim 12 or the vector of claim 13; and at least one of thetransposase DNA of claim 1 or 2, the vector of claim 6, the transposaseof claim 4, and the RNA of claim
 5. 17. The kit for geneticallymodifying mammals of claim 16, wherein a site to which a nucleic acidcan be inserted is provided in the nonautonomous transposon DNA of claim12, or in the region of the nonautonomous transposon DNA of claim 12 onthe vector of claim
 13. 18. A method for producing genetically modifiedmammalian cells, comprising the step of introducing the DNA of claim 8or the vector of claim 9 into mammalian cells.
 19. A method forproducing genetically modified mammalian cells, comprising the step ofintroducing mammalian cells with the nonautonomous transposon DNA ofclaim 12 or the vector of claim 13; and at least one of the transposaseDNA of claim 1 or 2, the vector of claim 6, the transposase of claim 4,and the RNA of claim
 5. 20. The method for producing geneticallymodified mammalian cells of claim 19, wherein a nucleic acid has beeninserted in the nonautonomous transposon DNA of claim 12, and the regionof the nonautonomous transposon DNA of claim 12 on the vector of claim13.
 21. A method for producing genetically modified mammals, comprisingthe step of injecting the DNA of claim 8 or the vector of claim 9 intononhuman mammals.
 22. A method for producing genetically modifiedmammals, comprising the step of introducing nonhuman mammals with thenonautonomous transposon DNA of claim 12 or the vector of claim 13; andat least one of the transposase DNA of claim 1 or 2, the vector of claim6, the transposase of claim 4, or the RNA of claim
 5. 23. A method forproducing genetically modified mammals, comprising the step generatingindividuals by mating a nonhuman mammal comprising the transposase DNAof claim 1 or 2 with a nonhuman mammal comprising the nonautonomoustransposon DNA of claim
 12. 24. A mammal for producing geneticallymodified nonhuman mammals, comprising the transposase DNA of claim 1 or2 or the nonautonomous transposon DNA of claim
 12. 25. The method forproducing genetically modified mammals of claim 22, wherein a nucleicacid has been inserted in to the nonautonomous transposon DNA of claim12, and the region of the nonautonomous transposon DNA of claim 12 inthe vector according to claim 13.